Although hyaluronan (HA), a large glycosaminoglycan (beta-1, 4-GlcA-Beta-1, 3-GlcNAc-]n, is expressed most abundantly in skin, its physiological functions remain largely unknown. Using a newly developed HA inhibitor "Pep-1", we observed that hapten-triggered migration of Langerhans cells (LC) can be blocked by Pep-1, providing the first evidence for the role of HA in LC homing. We also found that: a) dendritic cell (DC)-dependent T cell activation is blocked completely by Pep 1; b) DC express mRNAs for various HA synthases (Has1 to Has3) and hyaluronidases (Hyal-1 to Hyal-3) as well as HA polymers on their surfaces; and c) LC numbers are markedly diminished in Has 1/Has3-double knockout mice. Here we propose to define physiological roles of HA in regulating LC functions and to identify relevant HA receptors by focusing on CD44, receptor for HA-mediated motility (RHAMM), and lymphatic vessel endothelial HA receptor-i (LYVE-1) that is expressed selectively by lymphatic endothelial cells. Our specific aims are: 1) To study HA metabolism in DC. We will study de novo synthesis and surface expression of HA in DC isolated from Has1-/-, Has3-/-, and Has1/Has3-double knockout mice to identify the HA synthase(s) that control HA metabolism in DC. 2) To define functional roles played by two putative HA receptors in LC homing. We will study LC homing using CD44-/- RHAMM-/-, and CD44/RHAMM-double knockout mice. If DC isolated from these mice fail to achieve their homing in wild-type recipients, this will imply that CD44 and/or RHAMM expressed on DC act as homing receptors (Concept #1). 3) To determine whether HA polymers expressed on LC facilitate their homing. We will study the homing potentials of DC isolated from HA synthase deficient animals. If they fail to home in wild-type recipients, this will indicate that de novo synthesis and surface expression of HA in DC are required for their homing (Concept #2). We will identify HA receptors responsible for this function by using CD44/RHAMM-deficient animals as recipients. We will also construct LYVE-l-/- mice to determine whether LYVE-1 mediates the entry of migratory LC (expressing HA) in afferent lymphatics. 4) To study HA-mediated DC-T cell communication during antigen presentation. We will study T cell-stimulatory potentials of HA synthase-deficient DC to determine whether HA expressed on DC delivers co-stimulatory signals to T cells (Concept #3). We will also determine whether DC and/or T cells release intermediate HA fragments during antigen presentation and whether HA fragments trigger DC maturation (Concept #4). HA receptors responsible for these functions will be determined by testing HA-responsiveness of CD44/RHAMM-deficient T cells and DC. This study will provide new insights into LC biology and may lead to the development of new therapies for inflammatory skin disorders in which LC play pathogenic roles.